latrunculin-a and chelerythrine

Chemoattractant-stimulated pseudopod growth in human neutrophils was used as a model system to study the rate-limiting mechanism of cytoskeleton rearrangement induced by activated G-protein-coupled receptors. Cells were activated with N-formyl-Met-Leu-Phe, and the temperature dependence of the rate of pseudopod extension was measured in the presence of pharmacological inhibitors with known mechanisms of action. Three groups of inhibitors were used: (i) inhibitors sequestering substrates involved in F-actin polymerization (latrunculin A for G-actin and cytochalasin D for actin filament-free barbed ends) or sequestering secondary messengers (PIP-binding peptide for phosphoinositide lipids); (ii) competitively binding inhibitors (Akt-inhibitor for Akt/protein kinase B); and (iii) inhibitors that reduce enzyme activity (wortmannin for phosphoinositide 3-kinase and chelerythrine for protein kinase C). The experimental data are consistent with a model in which the relative involvement of a given pathway of F-actin polymerization to the measured rate of pseudopod extension is limited by a slowest (bottleneck) reaction in the cascade of reactions involved in the overall signaling pathway. The approach we developed was used to demonstrate that chemoattractant-induced pseudopod growth and mechanically stimulated cytoskeleton rearrangement are controlled by distinct pathways of F-actin polymerization.

Topics: Actins; Alkaloids; Androstadienes; Benzophenanthridines; Bridged Bicyclo Compounds, Heterocyclic; Cytochalasin D; Cytoskeleton; Enzyme Inhibitors; Enzymes; Humans; Kinetics; Lipids; Models, Chemical; N-Formylmethionine Leucyl-Phenylalanine; Neutrophils; Peptides; Phenanthridines; Phosphatidylinositol 3-Kinases; Protein Binding; Protein Kinase C; Receptors, G-Protein-Coupled; Temperature; Thiazoles; Thiazolidines; Time Factors; Wortmannin

The activated leukocyte cell adhesion molecule (ALCAM) is dynamically regulated by the actin cytoskeleton. In this study we explored the molecular mechanisms and signaling pathways underlying the cytoskeletal restraints of this homotypic adhesion molecule. We observed that ALCAM-mediated adhesion induced by cytoskeleton-disrupting agents is accompanied by activation of the small GTPases RhoA, Rac1 and Cdc42. Interestingly, unlike adhesion mediated by integrins or cadherins, ALCAM-mediated adhesion appears to be independent of Rho-like GTPase activity. By contrast, we demonstrated that protein kinase C (PKC) plays a major role in ALCAM-mediated adhesion. PKC inhibition by chelerythrine chloride and myristoylated PKC pseudosubstrate, as well as PKC downregulation by PMA strongly reduce cytoskeleton-dependent ALCAM-mediated adhesion. Since serine and threonine residues are dispensable for ALCAM-mediated adhesion and ALCAM is not phosphorylated, we can rule out that ALCAM itself is a direct PKC substrate. We conclude that PKCalpha plays a dominant role in cytoskeleton-dependent avidity modulation of ALCAM.

Topics: Activated-Leukocyte Cell Adhesion Molecule; Alkaloids; Antibodies, Monoclonal; Benzophenanthridines; Blotting, Western; Bridged Bicyclo Compounds, Heterocyclic; Cell Adhesion; Cytochalasin D; Cytoskeleton; Dose-Response Relationship, Drug; Down-Regulation; Fluorescein-5-isothiocyanate; Fluorescent Dyes; Humans; K562 Cells; Microscopy, Fluorescence; Phenanthridines; Phosphorus Radioisotopes; Protein Kinase C; Protein Kinase C-alpha; Retroviridae; rho GTP-Binding Proteins; Substrate Specificity; Tetradecanoylphorbol Acetate; Thiazoles; Thiazolidines

The eel intestinal epithelium responds to an acute hypertonic challenge by a biphasic increase of the rate of Cl(-) absorption (measured as short circuit current, Isc, and creating a negative transepithelial potential, V(te), at the basolateral side of the epithelium). While the first, transient phase is bumetanide-insensitive, the second, sustained phase is bumetanide-sensitive, reflecting activation of the apically located Na(+)-K(+)-2Cl(-) (NKCC) cotransporter, which correlates with the cellular RVI response. Here, we investigated the involvement of the cytoskeleton and of serine/threonine phosphorylation events in the osmotic stress-induced ion transport in the eel intestinal epithelium, focusing on the sustained RVI phase, as well as on the previously uncharacterized response to hypotonic stress. The study was carried out using confocal laser scanning microscopy, a quantitative F-actin assay, and transepithelial electrophysiological measurements (V(te) and Isc) in Ussing chambers. Hypertonic stress did not detectably alter either net F-actin content or F-actin organization. In contrast, a brief exposure to hypotonic stress decreased the total cellular F-actin content in eel intestinal epithelium by about 15%, detectable morphologically mainly as a decrease in the intensity of the apical brush border F-actin labeling.The bumetanide-sensitive response of V(te) and Isc to hypertonicity was potently inhibited by treatment with either cytochalasin, latrunculin A, colchicine, the protein kinase C (PKC) inhibitor chelerythrine, the myosin light chain kinase (MLCK) inhibitor ML-7, or the serine/threonine protein phosphatase inhibitor Calyculin A, but was unaffected by the PKA inhibitor H-89. The electrophysiological response of the epithelium to hypotonic stress was characterized by a sustained decrease of V(te) and Isc, which was smaller and recovered faster in the presence of either cytochalasin, latrunculin A, or colchicine. It is concluded that in eel intestinal epithelium, the changes in ion transport in response to both hyper- and hypotonic stress require the integrity of both F-actin and microtubules. In addition, the shrinkage-induced activation of NKCC appears to require the activity of both PKC and MLCK. It is suggested that NKCC regulation by hypertonic stress involves an interaction between the cytoskeleton and protein phosphorylation events.

Topics: Actins; Alkaloids; Anguilla; Animals; Azepines; Benzophenanthridines; Bridged Bicyclo Compounds, Heterocyclic; Bumetanide; Cell Size; Colchicine; Cytochalasins; Cytoskeleton; Enzyme Inhibitors; Hypertonic Solutions; Hypotonic Solutions; Intestinal Mucosa; Ion Transport; Isoquinolines; Marine Toxins; Microtubules; Naphthalenes; Osmotic Pressure; Oxazoles; Phenanthridines; Phosphorylation; Protein Serine-Threonine Kinases; Sulfonamides; Thiazoles; Thiazolidines